Method for composite cell-based implants

ABSTRACT

This invention is a method for the implantation of a combination of cells or cell-microcarrier aggregates wherein one component comprises a solid implantable construct and a second component comprises an injectable formulation. For example, in one embodiment, the solid implant may be first implanted to fill the majority of the cavity receiving the implant, and then cells or cell-microcarrier aggregates in an injectable format, with or without the addition of gelling materials to promote rapid gelling in situ, may be injected into spaces surrounding the solid implant in order to secure the solid implant in the site and/or to promote rapid adherence and/or integration of the solid implant to surrounding tissues. Also contemplated in this embodiment is that the cellular composition of the injectable component may differ from that of the solid component. For example, the solid implant may result from the culturing of chondrocytes on microcarriers or scaffolds, thereby resulting in an implant having cartilage-like properties, whereas the injectable cells or aggregates may result from the culturing of stem cells, resulting thereby in cells capable of producing cells of a chondrogenic, fibroblastic, myoblastic or osteoblastic phenotype. In this example, cells in the injectable aggregates may promote the fixation to or rapid integration of the solid cartilage implant into surrounding cartilage, connective tissue, muscle or bone, respectively.

RELATED APPLICATIONS

[0001] This application is a division of Ser. No. 09/922,909 filed Aug.6, 2001 which in turn was a continuation-in-part of application Ser. No.09/825,632 filed Apr. 4, 2001 for “Methods for FabricatingCell-Containing Implants”, which in turn is a continuation-in-part ofapplication Ser. No. 09/712,662 filed Nov. 14, 2000, which in turn is acontinuation-in-part of application Ser. No. 09/275,319 filed Mar. 24,1999, the contents of which prior applications are incorporated in theirentirety by reference herein.

FIELD OF THE INVENTION

[0002] The herein disclosed invention finds applicability in the fieldof preparation and implantation of tissue substitutes for tissuereplacement and for prosthesis.

BACKGROUND OF THE INVENTION

[0003] The present inventors have previously described a microcarrierspinner culture system that facilitated maintenance of chondrocyticphenotype while enhancing proliferation. Articular chondrocytes weregrown on dextran or crosslinked collagen microcarrier beads undercontrolled pH, oxygen levels, nutrient supply and mechanical agitationconditions. This represents a great advantage over the traditionalstatic monolayer culture system, which facilitates proliferation butleads to a fibroblastic shift in phenotype. Likewise, it offers analternative to the battery of three-dimensional gel or scaffold systems,which include agarose or collagen gels, calcium alginate gel, mixedfibrin-alginate gels, three-dimensional meshes of resorbable polymerssuch as polylactides or polyglycolides, and encapsulation in alginatebeads. These latter culture techniques facilitate the maintenance of achondrocytic phenotype, but are limited in maximizing proliferation.

[0004] A previously disclosed invention (referred to as PTO Ser. No.09/825,632) can be described as a method of preparing cells forimplantation comprising allowing cells (e.g., chondrocytes) to grow onmicrocarrier particles for an extended period of time and to secreteextracellular matrix components, thereby producing a cell-microcarrieraggregate useful for transplantation to a patient. The cell-microcarrieraggregates can be implanted directly or further cultured inside a moldthat has been shaped to configure the geometry of the area of the bodyreceiving the cells for transplantation. When further cultured in amold, cell-microcarrier aggregates are consolidated into an implantablestructure for repair or replacement of missing or diseased tissue. Themicrocarrier used to prepare the aggregate is a biocompatible,biodegradable material. This method also anticipates thatcell-microcarrier aggregates, or consolidated implants preparedtherefrom by further culturing in a mold, may be cryopreserved bystandard methods in order to maintain cell viability and aggregatestructure for future implantation or analysis.

[0005] In another embodiment of the PTO Ser. No. 09/825,632 invention,cell-microcarrier aggregates are cultured to provide a suspension ofindividual aggregates that may be implanted by injection by syringe orby other endoscopic or arthroscopic instruments suitable for theirimplantation into a diseased or damaged anatomic site. In thisembodiment, cell-microcarrier aggregates may be implanted without anyadditional material to bind the aggregates together after implantation.Alternatively, a material capable of polymerizing or gelling afterimplantation may be mixed with the aggregate suspension prior toimplantation in order to improve the fixation and localization of theaggregates after implantation, to stimulate more rapid consolidation ofthe aggregates in vivo, or to promote more rapid integration of theaggregates into the surrounding tissue.

[0006] Numerous studies of the implantation of solid tissue-engineeredimplants, especially soft-tissue analogs such as cartilage-likeimplants, have demonstrated that the fixation of these constructs tosurrounding tissues is a significant problem in the long-termlocalization of the construct at the implantation site and to thesubsequent integration of the implant with the surrounding tissue.Cartilage constructs such as those produced by culturing chondrocytes onbiodegradable scaffolds, for example, have been reported by researchersat MIT and Advanced Tissue Sciences to “seal off” around theirperipheral surfaces, resulting in inhibiting on blocking the integrationof these surfaces with surrounding articular cartilage followingimplantation. In particular, partial- or full-thickness defects inarticular cartilage are especially difficult to treat because mechanicalfixation by sutures results in further damage to the surroundingcartilage without resulting in firm fixation of the implant. The use oftissue glues such as fibrin or cyanoacrylate formulations, may result ininitial fixation of the implant, but may also inhibit or block thecellular processes leading to the bridging of the interface. Theinvention described herein provides a method for improving theintegration of such solid constructs into the surrounding host tissue byincorporating a suspension of cells or cell-microcarrier aggregates atthe interface of the implanted and surrounding host tissue, therebystimulating the integration of the implanted and host tissues.

PRIOR ART PATENTS

[0007] Masuda (U.S. Pat. No. 6,197,061) is for a method of preparing atransplantable cartilage matrix and its method of production. Autologouschondrocyte implantation is taught. Cell culture takes place in alginatebeads. Cell culture can take place over a period of 7 to 14 days orlonger.

[0008] Vacanti et al (U.S. Pat. No. 6,027,744) teach methods forgenerating new tissue using a hydrogel and tissue precursor cellsdelivered to a support and allowing the gel-cell composition to solidifywithin the support structure.

SUMMARY OF THE INVENTION

[0009] The herein disclosed invention proposes several alternativemethods for implanting tissue into a body cavity for the purpose ofrepairing the cavity. The methods employ in combination, for example, asolid aggregate of cells and microcarrier particles and a fluidcomposition of cells and microcarrier particles. In use the fluidcomposition serves to better fix the solid aggregate in the cavity to berepaired. These alternative methods are described in detail in thefigures and description set forth herein.

[0010] Definition of abbreviations used herein:

[0011] TGF-β—Transforming Growth Factor-β

[0012] BMP—Bone Morphogenetic Protein

[0013] PDGF—Platelet Derived Growth Factor

[0014] FGF—Fibroblast Growth Factor

DESCRIPTION OF THE INVENTION

[0015] Contemplated in this invention is the implantation of acombination of (1) cell-microcarrier aggregates or cell-scaffold orcell-free biomaterial formulations in a solid implantable format; and(2) cells or cell-microcarrier aggregates in an injectable format. Inthis context, a “solid” implant is a non-porous material that retainsits shape during handling. A solid implant may contain a high content ofwater if the water is substantially retained in the implant, such as incartilage or other connective tissue, for example. A solid implant maybe produced, for example, by culturing cells in a porous scaffold untilthe pores of the scaffold become filled with a tissue-like matrix. Forexample, in one embodiment, the solid implant may be first implanted tofill the majority of the cavity receiving the implant, and then asuspension of cells or of cell-microcarrier aggregates in the injectableformat, with or without the addition of gelling materials to promoterapid gelling in situ, may be injected into spaces surrounding the solidimplant or on the outer surface of the solid implant and surroundingtissue in order to fill the remaining space around the solid implantand/or to secure the solid implant in the site and/or to promote rapidadherence and/or integration of the solid implant and/or provide acovering for the implant to surrounding tissues. Alternatively, theinjectable formulation may first be applied to the site intended toreceive the implant, then the solid implant construct may be insertedinto the site. In this embodiment, the injectable formulation may serveto seat the solid construct into the defect, thereby helping to fix theimplant in place and to promote future integration of the implant withthe surrounding tissue(s). Finally, the injectable formulation may becoated onto the solid implant prior to implantation.

[0016] Also contemplated in this invention is that the cellularcomposition of the injectable component may differ from that of thesolid component. For example, the solid implant may result from theculturing of chondrocytes, thereby resulting in an implant havingcartilage properties, whereas the injectable aggregates may result fromthe culturing of stem cells, resulting thereby in cells having thecapability of producing cells of a fibroblastic, myoblastic orosteoblastic phenotype. In this example, cells in the injectableaggregates may promote the rapid integration of the solid cartilageimplant into surrounding soft tissue, muscle or bone, respectively.Cells are typically seeded onto the microcarriers at low density(1-4×10³ cells per cm²), and mixing the cells and microcarriers togetherfor periods sufficient for the cells to adhere to the microcarrier beads(2-4 hours). Microcarrier particles may be in the size range of100-500μ, with the preferred size predominantly in the range of100-400μ.

[0017] The microcarrier may be inorganic or organic resorbable materialssuitable for maintaining seeded cells in culture. Inorganic materialsinclude, for example: calcium phosphates, calcium carbonates, calciumsulfates or combinations of these materials. Organic materials mightinclude, for example: biopolymers such as collagen, gelatin, hyaluronicacid or chemically derived modifications of hyaluronic acid, chitin,chitosan or chitosan derivatives, fibrin, dextran, agarose, or calciumalginate, particles of tissue such as bone or demineralized bone,cartilage, tendon, ligament, fascia, intestinal mucosa or otherconnective tissues, or chemically modified derivatives of thesematerials. Organic materials might also include synthetic polymericmaterials, including, for example: polylactic acid, polyglycolic acid orcopolymers or combinations of the two, polyurethanes, polycarbonates,poly-caprolactones, hydrogels such as polyacrylates, polyvinyl alcohols,polyethylene glycols, or poly-ethyleneimines, or any other syntheticpolymers that can be produced in appropriate bead form.

[0018] In the production of an injectable formulation, cells orcell-microcarrier aggregates may be implanted without any additionalmaterial to bind the aggregates together after implantation.Alternatively, a material capable of polymerizing or gelling afterimplantation may be mixed with the aggregate suspension prior toimplantation in order to improve the fixation and localization of theaggregates after implantation, to stimulate more rapid consolidation ofthe aggregates in vivo, or to promote more rapid integration of theaggregates into the surrounding tissue. Examples of such bindingmaterials are blood, bone marrow, bone marrow concentrates, fibringlues, collagen, combinations of fibrin glues and collagen,transglutaminase-catalyzed binding systems, hyaluronic acid, calciumalginate gels, chitosan derivatives capable of gelling at bodytemperature, hydrogels such as polyacrylates, polyvinyl alcohols,polyethylene glycols, or polyethyleneimines, or similar materials withsuitable gelling compositions. In situ gelling of these materials may beinitiated by thermal, enzymatic or chemical catalysts, pH or ionicstrength changes or photo-initiation procedures.

[0019] Other bioactive factors, including, but not limited to, growthfactors, cytokines, antibodies, adhesion factors and intergrins, mayalso be incorporated into the injectable formulation to promote cellproliferation and/or differentiation, and/or improve fixation orintegration of the implants into the surrounding tissue(s). Such factorsmay include, for example, TGF-β, BMPs, PDGF, FGFs, interleukins and thelike.

[0020] Although, the herein disclosed invention has been characterizedas using chondrocytes, it may be embodied using any cells that secreteextracellular matrix components suitable for causing the cells ormicrocarrier-cell suspension to aggregate or to adhere to surroundingtissue in suspension culture, in suitable molding devices, or in situfollowing injection into a body cavity or tissue. Such cells may alsoinclude, for example, osteoblasts, myoblasts, keratinocytes, fibroblastssuch as those harvested from tendon, ligament, skin, meniscus or disk ofthe temporomandibular joint or intervertebral joint, or multi-potentstem cells that are capable of differentiating into matrix-producingcells, including mesenchymal stem cells, pluripotent stem cells frommuscle, fat or skin, or embryonic stem cells.

[0021] The herein disclosed invention also contemplates the formation ofimplants using a combination of one or more consolidated solid implantsand an injectable cell or cell-microcarrier aggregate component capableof filling voids surrounding the solid implants or covering the implantor covering the implant and surrounding tissue and thereby, for example,to secure the solid implant in the site and/or to promote rapidadherence and/or integration of the solid implant to surroundingtissues. In this embodiment, the solid implants and injectable cells oraggregates may be derived from different cells, thereby promoting rapidintegration in a specific surrounding tissue such as cartilage, muscle,bone, skin, tendon or ligament.

[0022] The herein disclosed invention is related to, and can be usedwith, a co-pending application (owned by a common assignee) and in whichat least one of the present inventors is a joint inventor.

[0023] This pending application, identified by PTO Ser. No. 09/825,632,is directed to a method of replacing a tissue or body part or filling avoid in head and neck area comprising the steps of obtaining anon-diseased cell sample from the respective patient's head and neckarea, rapidly growing additional cells in a bioreactor and within apredetermined mold or culture chamber which is the mirror image of thepatient's tissue, body part or void, such that a molded tissue or bodypart is produced, and surgically implanting the molded tissue or bodypart as a replacement in the patient's head and neck area, such that themolded tissue or body part regenerates therein and may thereby fuse withthe adjacent tissues in the head and neck area of the respectivepatient. The method also involves obtaining cells from the nasal areawhich may be chondrocytes. The method can include a scaffold made from abiodegradable microcarrier material for supporting the molded tissue orbody part. In a more general embodiment of the invention, cells may beobtained from any anatomic location of the patient to receive theimplant or from another human donor, and the resulting material producedby the culture method may be implanted at any location requiring theimplant.

[0024] Also contemplated in this invention is a kit comprising theimplant material and a means for implanting the implant into the desiredanatomic site. For example, the kit may comprise a fluid suspension ofcell-microcarrier aggregates, as well as, a syringe, arthroscopic deviceor endoscopic device used for injecting the suspension into the desiredanatomic site. Alternatively, the kit may comprise a molded implantformed by further culturing the cell-microcarrier aggregates to form asolid device, along with tools for further shaping the implantintraoperatively, and tools or materials for implanting the implant byopen or minimally invasive surgical procedures.

[0025] Advantages of the method. The invention comprises the use of twoformulation strategies for implantation of cells or cell-microcarrieraggregates, one as an injectable dispersion of cells or aggregates and asecond formulation as a solid, semi-solid molded or formed structure ofconsolidated aggregates prepared by further culturing of aggregates in amold device. This method results in an implantation procedure that fillsthe majority of a tissue-requiring site with an approximately-shapedsolid implant and permits the remainder of the tissue-requiring site tobe filled with an injectable formulation of cells or cell-microcarrieraggregates that may conform to the irregular shape of the site. Theinvention further provides a method to improve fixation or localizationof a solid implant and/or to promote its more rapid integration intosurrounding tissues, wherein the second implant comprising injectablecells or aggregates may be used to form or initiate a bond between thesolid implant and the surrounding tissue.

WITH REFERENCE TO THE DRAWINGS

[0026] FIGS. 1-6 are schematic representations of applying cellaggregates to fill a body cavity.

[0027] In the block diagram of FIG. 2, corresponding to FIGS. 1A-1D,respectively, an aggregate of cells and microcarrier particles 10 areinserted into the body cavity 11, corresponding to (FIG. 1A and FIG.1B); and additional cells on microcarrier particles in the fluid state(denoted by 12) are injected between the surface of the body cavity 11and the cell-microcarrier particle aggregate 10 (FIG. 1C and FIG. 1D)via a syringe 14 to assure an integral fit between the cell aggregate 10and the tissue 13. Of course, the microcarrier particles in the fluidstate could be applied over the aggregate of cells and microcarrierparticles.

[0028] In the block diagram of FIG. 4, FIGS. 3A-3D, respectively, cellson microcarrier particles in the fluid state 12 are first injected bythe syringe 14 to coat the surface of the body cavity 11 (FIGS. 3A and3B); and then the molded aggregate of cells on the microcarrierparticles 10 are inserted (FIG. 3C) to form the finished implant (FIG.3D).

[0029] With reference to the block diagram of FIG. 6, corresponding toFIGS. 5A-5D, respectively, cells on microcarrier particles in the fluidstate 12 are coated by the syringe 14 onto a formed aggregation of cells10 which approximate the body cavity 11 (FIGS. 5A and 5B); and then thecoated formed aggregation of cells 10 is implanted into the body cavity11 (FIGS. 5C and 5D).

[0030] With reference to FIG. 7, there is shown a flow-chart ofalternative methods of implanting aggregates of cells on microcarrierparticles into a body cavity.

EXAMPLES Example 1—Formation of Solid Tissue Implants

[0031] In Example 1 of PTO Ser. No. 09/825,632, one of the presentinventors demonstrated that nasal chondrocytes propagated inmicrocarrier spinner culture would proliferate and produce extracellularmatrix components similar to that produced by articular chondrocytes.Cartilage was obtained from five patients during nasal septumreconstruction. Chondrocytes isolated by collagenase digestion weredirectly seeded at 4×10³ cells/cm² onto Cellagen microcarriers (100-400cm², derived from bovine corium, (ICN, Cleveland, Ohio)) or in monolayerculture. Monolayer and microcarrier spinner cultures were incubated at37E C, 5% CO₂ for fourteen days. Chondrocytes were harvested and cellsamples enumerated in trypan blue vital dye. To analyze for proteoglycanproduction, cells were pulsed for 60 hours with 50 iCi/ml, ³⁵SO₄. Theproteoglycans were extracted using 4M guanidinium HCl for 24 hours at 4°C. and radiolabeled incorporation was determined by liquid scintillationcounting. Aliquots were electrophoresed on 0.6% agrose-1.25polyacrylamide gels and then autoradiographed. The Dc protein assay fromBioRad was used to assess protein concentration in the cell-associatedfractions (CAF). The protein concentration in the CAF was used tonormalize the total CPM in each fraction. Replicates of the cells werefrozen for subsequent RNA isolation. Gene Markers were determined usingRT-PCR.

[0032] Chondrocytes isolated from nasal cartilage proliferated inmicrocarrier spinner culture within two weeks producingcell-microcarrier aggregates with cartilage-like extracellular matrix.Cell numbers increased up to 17-fold. Cell-microcarrier aggregatesfurther cultured began to further aggregate into a cartilage-likematerial that was produced within thirty days. Chondrocytes expressedcollagen type II and aggrecan but not collagen type I. Propagation ofchondrocytes from this cartilage site in spinner culture maintained theexpression of collagen type II while decreasing the expression ofcollagen type I. The newly synthesized proteoglycans appeared to have ahigh molecular weight. Histology indicated a tissue morphologyconsistent with that of hyaline cartilage.

[0033] This example demonstrates that nasal chondrocytes multiply inCellagen-microcarrier spinner culture. Thus, chondrocytes retrieved froma non-articulating cartilaginous site is able to maintain features oftheir original phenotype. After 30 days in culture, cell-microcarrierconstructs had aggregated to form consolidated structures with hyalinecartilage-like properties. These aggregated materials would be suitablefor implantation, or the cell-microcarrier aggregates formed after 7-14days may be transferred to an Implant Assembly Unit to form a solidimplant with a specific geometric shape. The construct formed by thisprocess illustrates one type of solid formulation that may be implantedaccording to the instant invention. Alternatively, solid formulation mayformed by the extended culturing of chondrocytes or stem cells, forexample, on porous, biocompatible solid scaffolds suitable forimplantation into the body. Such solid formulations have been describedfrom many research groups including researchers at MIT, Advanced TissueSciences, Case Western Reserve University, Osiris Therapeutics andothers.

Example 2—Formation of Injectable Tissue Implants

[0034] The cell-seeded microcarriers described in Example 1 aremaintained in spinner culture at 60 rpm, 37° C., 5% CO₂ for 14 to 21days to allow visible secretion of extracellular matrix to take place inenriched medium (Dulbecco essential media containing NCTC-109, OPI(oxaloacetate, pyruvate, insulin), 1-glutamine, gentamycin, and fetalcalf serum). The cell-microcarrier aggregates are subsequentlycentrifuged at 200 g for 15 minutes and 4° C. The supernatant fluid isremoved and the aggregates are resuspended in a fluid medium, such asphosphate buffered saline solution, suitable for injection into thebody. The resuspended aggregates are next transferred to a syringe orother suitable implantation device whereby the suspension is implanteddirectly into the anatomic site or cavity requiring the cartilageimplant. Alternatively, the cell-microcarrier aggregates may becryopreserved and thawed prior to injection.

[0035] This example illustrates one method for producing an injectablecell-microcarrier aggregate suspension suitable as the injectableformulation of the instant invention. Alternatively, appropriate cellsmay be grown in spinner or monolayer culture, harvested by enzymatic(collagenase) treatment and concentrated to an injectable suspension.

Example 3—Formation of Injectable Tissue Implants Containing aGel-forming System

[0036] The cell-seeded microcarriers of Example 1 are maintained inspinner culture at 60 rpm, 37° C., 5% CO₂ for 14 to 21 days to allowvisible secretion of extracellular matrix to take place in enrichedmedium (Dulbecco essential media containing NCTC-109, OPI (oxaloacetate,pyruvate, insulin), 1-glutamine, gentamycin, and fetal calf serum). Thecell-microcarrier aggregates are subsequently centrifuged at 200 g for15 minutes and 4° C. The supernatant fluid is removed and the aggregatesare resuspended in a fluid medium, such as phosphate buffered salinesolution, suitable for injection into the body. In this example,fibrinogen solution is added in concentration sufficient to form a gelin vivo, and thrombin is added in sufficient concentration to cause theformation of a fibrin gel in 5-10 minutes in vivo. The resuspendedaggregates are next transferred to a syringe or other suitableimplantation device whereby the suspension is implanted directly intothe anatomic site or cavity requiring the cartilage implant. Rapidgelation of the fibrinogen/thrombin system promotes formation of afibrin gel in situ that stabilizes the localization of the injectedaggregates, yet permits continued secretion of extracellular matrixcomponents. During the next 14-28 days, or after a suitable perioddepending on the site and the rate of degradation of the fibrin gel, thecells continue to secrete extracellular matrix, thereby consolidatingthe cell-microcarrier suspension into a solid cartilage-like mass.

[0037] This example illustrates one method for producing an injectablecell-microcarrier aggregate suspension containing a gel-forming systemsuitable as the injectable formulation of the instant invention.Alternatively, other compositions may be used, such collagen,combinations of fibrin/collagen, transglutaminase-catalyzed bindingsystems, hyaluronic acid, calcium alginate gels, chitosan derivativescapable of gelling at body temperature, hydrogels such as polyacrylates,polyvinyl alcohols, polyethylene glycols, or polyethyleneimines, orsimilar materials with suitable gelling compositions. In situ gelling ofthese materials may be initiated by thermal, enzymatic or chemicalcatalysts, pH or ionic strength changes or photo-initiation procedures.

Example 4—Implantation of Composite Cell-based Implants intoCraniofacial Tissues

[0038] A solid implant construct comprising autologous nasal septalchondrocytes on bioresorbable polylactic acid microcarrier beads isproduced by the methods described in Example 1.

[0039] An injectable implant formulation comprising nasal septalchondrocytes on bioresorbable polylactic acid microcarrier beads isproduced by the methods described in Example 2.

[0040] A defect in a human nose or ear is filled by a combination of thesolid implant and the injectable formulation by the following procedure:

[0041] 1. The lesion is exposed and prepared for receiving the implantby cleaning, debriding and/or modifying the defect site to form a shapesuitable for receiving the implant;

[0042] 2. The solid implant is cut to the approximate size and shape ofthe lesion and then placed into the lesion site;

[0043] 3. A syringe containing the injectable formulation of cells orcell-microcarrier aggregates is used to deliver the suspension into thespaces surrounding the solid implant;

[0044] 4. The incision is closed by sutures.

[0045] This example illustrates a method for implanting a composite of asolid implant and an injectable formulation of cells orcell-microcarrier aggregates to reconstruct a defect in craniofacialtissue.

Example 5—Implantation of Composite Cell-based Implants in ArticularJoints

[0046] A solid implant construct comprising autologous articularchondrocytes on a bioresorbable polylactic acid scaffold is produced bythe methods described in Example 1.

[0047] An injectable implant formulation comprising allogeneicmesenchymal stem cells in an injectable phosphate buffered salinesolution containing fibrinogen and thrombin is produced by the methodsdescribed in Example 3.

[0048] A partial thickness lesion in the articular cartilage of a kneeis filled by a combination of the solid implant and the injectableformulation by the following procedure:

[0049] 1. The lesion is evaluated by arthroscopic methods and preparedfor receiving the implant by cleaning, debriding and/or modifying thedefect site to form a shape suitable for receiving the implant;

[0050] 2. A arthroscopic delivery tool containing the injectableformulation of cells is used to coat the surface of the lesion with theformulation;

[0051] 3. The solid implant is cut to the approximate size and shape ofthe lesion, arthroscopically placed into the lesion site, observed untilthe fibrin glue sets;

[0052] 4. The arthroscopic tools are removed and incisions closed bystandard methods.

[0053] This example illustrates a method for implanting a composite of asolid implant by using an injectable formulation of cells in a fibringlue to fix the solid implant to the lesion site and to promote rapidintegration of the solid implant construct into the surroundingcartilage tissue.

[0054] Obviously, many modifications may be made without departing fromthe basic spirit of the present invention. Accordingly, it will beappreciated by those skilled in the art that within the scope of theappended claims, the invention may be practiced other than has beenspecifically described herein.

We claim:
 1. A method of treating a lesion or cavity in a tissuecomprising filling said lesion or cavity with a solid implant along withan injectable cell-containing formulation.
 2. The method of claim 1wherein the solid implant contains cells.
 3. The method of claim 1wherein the solid implant and injectable formulation are implanted intoa cavity or an anatomic site requiring repair or replacement of missingor diseased tissue.
 4. The method of claim 2 wherein the cells in thesolid implant are chondrocytes.
 5. The method of claim 2 wherein thecells are extracellular matrix producing cells selected fromchondrocytes; osteoblasts; keratinocytes; fibroblasts derived from skin,tendon, ligament, meniscus, temporalmandibular joint or intervertebraljoint, disk or any other connective tissue; stem cells derived fromskin, tendon, ligament, meniscus, disk or any other connective tissue;stem cells derived from bone marrow stroma, muscle, skin or other stemcell-containing tissue; embryonic stem cells; or combinations of thesecells that may be seeded onto the microcarrier.
 6. The method of claim 2wherein the solid implant is made by culturing cells on biodegradablemicrocarriers.
 7. The method of claim 6 wherein the microcarrier isselected from inorganic materials selected from calcium phosphates,calcium carbonates, calcium sulfates or combinations of these materials;organic materials including biopolymers; synthetic polymeric materials;particles of tissues; or chemically modified derivatives of thesematerials.
 8. The method of claim 7 wherein the microcarrier is selectedfrom inorganic materials selected from calcium phosphates, calciumcarbonates, calcium sulfates or combinations of these materials; organicmaterials including biopolymers selected from collagen, gelatin,hyaluronic acid or chemically derived modifications of hyaluronic acid,chitin, chitosan or chitosan derivatives, fibrin, dextran, agarose, orcalcium alginate, or synthetic polymeric materials selected frompolylactic acid, polyglycolic acid or copolymers or combinations of thetwo, polyurethanes, polycarbonates, polycaprolactones, hydrogels such aspolyacrylates, polyvinyl alcohols, polyethylene glycols, orpolyethyleneimines; or particles of tissues selected from bone ordemineralized bone, cartilage, tendon, ligament, fascia, intestinalmucosa or other connective tissues, or chemically modified derivativesof these materials.
 9. The method of claim 6 wherein cell-microcarrieraggregates are further cultured inside a mold which has been shaped toconfigure the geometry of the area of the body receiving the cells forimplantation.
 10. The method of claim 6 wherein the mold is shaped toproduce a form that can be cut or modified to a desired shape at thetime of implantation.
 11. The method of claim 6 wherein the culturing ofsaid cells takes place over one to five weeks.
 12. The method of claim 6wherein the cells are seeded onto the microcarrier at a density of 1 to4×10³ cells/cm².
 13. The method of claim 1 wherein the solid implant ismade by culturing cells on a porous biodegradable scaffold.
 14. Themethod of claim 13 wherein the scaffold is selected from inorganicmaterials, organic materials including biopolymers, synthetic polymericmaterials or particles of tissues.
 15. The method of claim 13 whereinthe scaffold is selected from inorganic materials selected from calciumphosphates, calcium carbonates, calcium sulfates or combinations ofthese materials; organic materials including biopolymers selected fromcollagen, gelatin, hyaluronic acid or chemically derived modificationsof hyaluronic acid, chitin, chitosan or chitosan derivatives, fibrin,dextran, agarose, or calcium alginate, or synthetic polymeric materialsselected from polylactic acid, polyglycolic acid or copolymers orcombinations of the two, polyurethanes, polycarbonates,polycaprolactones, hydrogels selected from polyacrylates, polyvinylalcohols, polyethylene glycols, or polyethyleneimines; or particles oftissues selected from bone or demineralized bone, cartilage, tendon,ligament, fascia, intestinal mucosa or other connective tissues, orchemically modified derivatives of these materials.
 16. The method ofclaim 13 wherein the scaffold has been shaped to configure the geometryof the area of the body receiving the cells for implantation.
 17. Themethod of claim 13 wherein the scaffold is shaped to produce a form thatcan be cut or modified to a desired shape at the time of implantation.18. The method of claim 1 wherein the solid implant is a biodegradablescaffold.
 19. The method of claim 18 wherein the scaffold is selectedfrom inorganic materials, organic materials, including biopolymers,synthetic polymeric materials, particles of tissues, or chemicallymodified derivatives of these materials.
 20. The method of claim 18wherein the scaffold is selected from inorganic materials selected fromcalcium phosphates, calcium carbonates, calcium sulfates or combinationsof these materials; or organic materials including biopolymers selectedfrom collagen, gelatin, hyaluronic acid or chemically derivedmodifications of hyaluronic acid, chitin, chitosan or chitosanderivatives, fibrin, dextran, agarose, or calcium alginate; or syntheticpolymeric materials such as polylactic acid, polyglycolic acid orcopolymers or combinations of the two, polyurethanes, polycarbonates,polycaprolactones, hydrogels such as polyacrylates, polyvinyl alcohols,polyethylene glycols, or polyethyleneimines; or particles of tissuesselected from bone or demineralized bone, cartilage, tendon, ligament,fascia, intestinal mucosa or other connective tissues, or chemicallymodified derivatives of these materials.
 21. The method of claim 18wherein the scaffold has been shaped to configure the geometry of thearea of the body receiving the cells for implantation.
 22. The method ofclaim 18 wherein the scaffold is shaped to produce a form that can becut or modified to a desired shape at the time of implantation.
 23. Themethod of claim 1 wherein the cells in the injectable formulation arechondrocytes.
 24. The method of claim 1 wherein the cells in theinjectable formulation are extracellular matrix producing cells selectedfrom chondrocytes; osteoblasts; keratinocytes; fibroblasts derived fromskin, tendon, ligament, meniscus, temporalmandibular joint orintervertebral joint, disk or any other connective tissue; stem cellsderived from bone marrow stroma, muscle, skin or other stemcell-containing tissue; embryonic stem cells; or combinations of thesecells that may be seeded onto the microcarrier.
 25. The method of claim1 wherein the injectable formulation is made by cells in a solutionsuitable for injection.
 26. The method of claim 1 wherein the injectableformulation is made by culturing cells on biodegradable microcarriers.27. The method of claim 26 wherein the microcarrier is selected frominorganic materials, organic materials including biopolymers, syntheticpolymeric materials, or chemically modified derivatives of thesematerials.
 28. The method of claim 26 wherein the microcarrier isselected from inorganic materials selected from calcium phosphates,calcium carbonates, calcium sulfates or combinations of these materials;organic materials including biopolymers selected from collagen, gelatin,hyaluronic acid or chemically derived modifications of hyaluronic acid,chitin, chitosan or chitosan derivatives, fibrin, dextran, agarose, orcalcium alginate; synthetic polymeric materials such as polylactic acid,polyglycolic acid or copolymers or combinations of the two,polyurethanes, polycarbonates, polycaprolactones, hydrogels selectedfrom polyacrylates, polyvinyl alcohols, polyethylene glycols, orpolyethyleneimines; or particles of tissues such as bone ordemineralized bone, cartilage, tendon, ligament, fascia, intestinalmucosa or other connective tissues, or chemically modified derivativesof these materials.
 29. The method of claim 26 wherein the injectableformulation is a fluid medium suitable for injection selected from:isotonic saline for injection, phosphate buffered saline, or Hank'sbalanced salt solution.
 30. The method of claim 26 wherein the fluidmedium suitable for injection contains a material capable ofpolymerizing or gelling after implantation.
 31. The method of claim 30wherein the material is selected from fibrin glues, collagen,combinations of fibrin/collagen, hyaluronic acid, calcium alginate gels,chitosan derivatives capable of gelling at body temperature, hydrogelssuch as polyacrylates, polyvinyl alcohols, polyethylene glycols, orpolyethyleneimines.
 32. The method of claim 26 wherein the fluid mediumsuitable for injection contains a bioactive factor.
 33. The method ofclaim 32 wherein the bioactive factor is selected from cytokines, growthfactors, antibodies, adhesion factors or integrins.
 34. The method ofclaim 32 wherein the bioactive factor is selected from TGF-β, BMPs,PDGF, FGFs and interleukins.
 35. The method of claim 30 wherein the insitu gelling of these materials is initiated by thermal, enzymatic orchemical catalysts, pH or ionic strength changes or photo-initiationprocedures.
 36. A method for replacing a tissue or body part or fillinga void in a tissue comprising (1) preparing a solid implant; (2)preparing an injectable cell-containing formulation; (3) implanting thesolid implant into a cavity or defect in the tissue; and (4) injectingthe injectable cell-containing formulation into the interstices betweenthe tissue and the solid implant.
 37. The method of claim 36 in whichthe solid implant contains cells.
 38. The method of claim 36 wherein thecells are chondrocytes.
 39. The method of claim 36 wherein the cells areselected from chondrocytes; osteoblasts; keratinocytes, fibroblastsderived from skin, tendon, ligament, meniscus, disk or any otherconnective tissue; stem cells derived from bone marrow stroma, muscle,skin or other stem cell-containing tissue; embryonic stem cells; orcombinations of these cells that may be seeded onto the microcarrier.40. The method of claim 37 wherein the cells used to prepared the solidimplant differ from the cells used to prepare the injectablecell-containing formulation.
 41. The method of claim 37 wherein thesolid cell-containing implant is prepared by culturing cells on a solidscaffold.
 42. The method of claim 37 wherein the solid cell-containingimplant is prepared by culturing cells on microcarrier particles. 43.The method of claim 36 wherein the injectable cell-containingformulation is prepared by culturing cells on microcarrier particles.44. The method of claim 36 wherein the injectable cell-containingformulation comprises a suspension of cells in a medium suitable forinjection.
 45. A method for replacing a tissue or body part or filling avoid in a tissue comprising (1) preparing a solid cell-containingimplant from cells; (2) preparing an injectable cell-containingformulation of cell-microcarrier aggregates; (3) coating the surface ofthe cavity or lesion of the tissue with the injectable cell-containingformulation; and (4) implanting the solid cell-containing implant intothe cavity or lesion such that it is in contact with the injectablecell-containing formulation.
 46. The method of claim 45 wherein thecells are chondrocytes.
 47. The method of claim 45 wherein the cells areselected from chondrocytes; osteoblasts; fibroblasts derived from skin,tendon, ligament, meniscus, disk or any other connective tissue; stemcells derived from bone marrow stroma, muscle, skin or other stemcell-containing tissue; embryonic stem cells; or combinations of thesecells that may be seeded onto the microcarrier.
 48. The method of claim45 wherein the cells used to prepared the solid implant differ from thecells used to prepare the injectable cell-containing formulation. 49.The method of claim 45 wherein the solid cell-containing implant isprepared by culturing cells on a solid scaffold.
 50. The method of claim45 wherein the solid cell-containing implant is prepared by culturingcells on microcarrier particles.
 51. The method of claim 45 wherein theinjectable cell-containing formulation is prepared by culturing cells onmicrocarrier particles.
 52. The method of claim 45 wherein theinjectable cell-containing formulation comprises a suspension of cellsin a medium suitable for injection.
 53. The method of replacing a tissueor body part or filling a void in the head or neck area comprising thesteps of obtaining a non-diseased, cell sample from the respectivepatient's head and neck area, rapidly growing additional cells obtainedfrom said cell sample in a bioreactor to produce a suspension ofcell-microcarrier aggregates, and further culturing some of thecell-microcarrier aggregates within a predetermined mold which is themirror image of the patient's tissue, body part or void, such that amolded tissue or body part is produced, and surgically implanting themolded tissue or body part in combination with an injectable suspensionof cell-microcarrier aggregates as a replacement in the patient's headand neck area, such that the implanted tissues regenerates therein andfuses with the adjacent tissues in the head and neck area of therespective patient.
 54. An implant for a cavity in the body of apatient, comprising a formed aggregation of cells on first microcarrierparticles which approximates the size and shape of the cavity in thepatient's body, and an interface layer of cells between the formedaggregation of cells and the cavity in the patient's body, wherein theformed aggregation of cells are implanted in a substantially solid formfollowing culturing of the cells on the micro carrier particles during afirst time period, and wherein the interface layer of cells have beencultured on second microcarrier particles during a second time periodwhich is substantially shorter than the first time period and have beenapplied while in a substantially fluid state.
 55. The implant of claim54, wherein the cells on the formed aggregation of cells compriseschondrocytes, thereby resulting in an implant having cartilageproperties.
 56. The implant of claim 55, wherein the formed aggregationof cells comprises a molded aggregation of cells.
 57. The implant ofclaim 54, wherein the interface layer of cells comprises cultured stemcells, thereby promoting the rapid integration of the formed aggregationof cells into the soft tissue, muscle or bone surrounding the bodycavity.
 58. The implant of claim 57, wherein the cultured stem cells arecapable of producing cells selected from the group consisting offibroblastic, myoblastic or osteoblastic phenotype cells.
 59. Theimplant of claim 54, wherein the interface layer of cells is injectedinto the body cavity prior to the implantation of the formed aggregationof cells therein.
 60. The implant of claim 54, wherein the interfacelayer of cells is injected into the body cavity after the implantationof the formed aggregation of cells therein.
 61. The implant of claim 54,wherein the interface layer of cells is coated onto the formedaggregation of cells, and then the coated formed aggregation of cells isimplanted into the body cavity.
 62. The method of making an implant forinsertion into the body cavity of a patient, comprising the steps offorming a sintered aggregation of cells cultured on first microcarrierparticles during a first time period, such that the sintered aggregationis in a substantially solid state, forming a plurality of cells onsecond microcarrier particles during a second time period which isshorter than the first time period, such that the plurality of cells onthe second microcarrier particles is in a substantially fluid state, andcombining the sintered aggregation of cells and the substantially-fluidplurality of cells to make the implant.
 63. The method of claim 62,wherein the sintered aggregation of cells approximates the size andshape of the body cavity, and wherein the substantially-fluid pluralityof cells forms an interface layer between the body cavity and thesintered aggregation of cells.
 64. The method of claim 63, wherein thesubstantially-fluid plurality of cells is injected into the body cavityafter the sintered aggregation of cells is inserted into the bodycavity.
 65. The method of claim 63, wherein the substantially-fluidplurality of cells is injected into the body cavity before the sinteredaggregation of cells is inserted into the body cavity.
 66. The method ofclaim 63, wherein the substantially-fluid plurality of cells forms acoating on the sintered aggregation of cells before the sinteredaggregation of cells is inserted into the body cavity.
 67. The method ofclaim 62, wherein the cells comprise chondrocytes.
 68. The method ofclaim 62 wherein the cells are selected from chondrocytes; osteoblasts;fibroblasts derived from skin, tendon, ligament, meniscus, disk or anyother connective tissue; stem cells derived from bone marrow stroma,muscle, skin or other stem cell-containing tissue; embryonic stem cells;or combinations of these cells that may be seeded onto the microcarrier.69. The method of claim 62 wherein the microcarrier is selected frominorganic materials selected from calcium phosphates, calciumcarbonates, calcium sulfates or combinations of these materials; organicmaterials including biopolymers selected from collagen, gelatin,hyaluronic acid or chemically derived modifications of hyaluronic acid,chitin, chitosan or chitosan derivatives, fibrin, dextran, agarose, orcalcium alginate; synthetic polymeric materials such as polylactic acid,polyglycolic acid or copolymers or combinations of the two,polyurethanes, polycarbonates, polycaprolactones, hydrogels selectedfrom polyacrylates, polyvinyl alcohols, polyethylene glycols, orpolyethyleneimines; or particles of tissues such as bone ordemineralized bone, cartilage, tendon, ligament, fascia, intestinalmucosa or other connective tissues, or chemically modified derivativesof these materials.
 70. Method of treating a lesion on or in the skincomprising filling said lesion with a solid cell-containing implantalong with an injectable cell-containing formulation.